You have just landed your dream job as a postdoctoral fellow in a famous C. eleg

Question

You have just landed your dream job as a postdoctoral fellow in a famous C. elegans lab. Your boss hands you a plate of hlh-6 mutant worms and tells you to figure out the function of hlh-6. He tells you that hlh-6 mutant worms have fewer neurons than wild type controls but nothing else is known about the mutant, other than which gene is affected. Your first approach is to look if any hlh-6 orthologs exist in other species to get a potential clue about hlh-6 function.

Design a set of experiments to ascertain whether or not the function of the human hlh-6 ortholog is conserved with C. elegans. Be sure to include the appropriate controls for your experiment.

Explanation / Answer

A single immunoglobulin domain protein required for the localization of acetylcholine receptors at the C.elegans neuromuscular junction. Acetylcholine receptors sensitive to the nematode-specific cholinergic agonist levamisole (L-AChRs) form clusters at the C. elegans neuromuscular junctions (NMJs) and their proper localization is critical for efficient excitatory neurotransmission. Here we demonstrate that one Ig is a new gene required for L-AChR synaptic clustering. We have previously shown that L-AChR clustering requires two proteins, LEV-9 and LEV-10, which form an extracellular synaptic scaffold. LEV-9, a CCP-domain rich secreted protein, physically interacts with the ectodomain of LEV 10, a CUB-domain rich type 1 transmembrane protein. Interestingly, none of the proteins of the L-AChR/LEV-9/LEV-10 complex forms clusters at the synapse by itself, indicating that additional components are necessary for synaptic localization of this complex. lev-10 and lev-9 mutants show partial resistance to levamisole: they sense levamisole and hypercontract but do not die on drug concentrations lethal for wild-type animals. To identify new components of the L-AChR clustering machinery we performed a screen for mutants with partial resistance to levamisole after EMS mutagenesis and we isolated a new mutant allele, kr39 . Using classical genetic mapping we identified a mis-sense point mutation in the predicted gene oig-4. We isolated a second oig-4 mutant allele in a non-complementation screen. In both mutants, levamisole-resistance is rescued by providing an oig-4 genomic fragment. Immunostaining of L-AChR subunits and in vivo imaging of knock-in strains with fluorescent L-AChR subunits demonstrate that L-AChRs do not localize properly at the NMJs of oig-4 mutants. Moreover, the LEV-10 protein is no longer detected at synapses of these mutants. However, presynaptic boutons are properly formed. OIG-4 is specifically required for the L-AChR clustering as the UNC-49 GABA receptor and the ACR-16 N-AChRs are clustered normally at NMJs of oig-4 mutants.

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